The need for rapid and sensitive methods to detect drug-resistant tuberculosis (TB) has never been greater. The incidence of multi-drug-resistant (MDR) and extensively-drug resistant (XDR) TB is increasing at an alarming rate. Even in the absence of MDR and XDR TB, rapid Fluoroquinolone (FQ) resistance testing will be increasingly needed for patients who are treated with the Moxifloxacin-based regimes that are expected to partially replace Isoniazid (INH)-based treatment in the future. Our group developed the Xpert MTB/RIF (Xpert) assay, the first ever self-contained, near-patient assay that can perform TB detection and also detect resistance to the drug Rifampicin (RIF). This test is transforming TB detection worldwide by making TB detection simple and rapid. However in regards to drug resistance detection, the Xpert assay is limited to identifying patients who potentially have MDR by using RIF-resistance as a surrogate. The Xpert assay cannot tell whether a patient with RIF resistance remains INH-susceptible, and thus could still be treated with an INH-containing regime. Nor can it identify which RIF-resistant patients can be treated with a FQ and/or aminoglycoside as opposed to those who have XDR TB. Thus, patients are at risk for being either over treated or undertreated, with potentially serious consequences. Furthermore, patients with undiagnosed XDR can remain infectious for prolonged periods, further spreading their disease. Here, we propose to combine highly innovative 10-color real-time PCR detection (that makes use of novel large Stokes-shift dyes), innovative three phase PCR fluidics, sloppy molecular beacon probes, the existing GeneXpert platform and basic Xpert assay cartridge to create a new assay that identifies resistance to INH, the FQs, amikacin and kanamycin, important types of resistance not detected by the Xpert assay. This new Xtend-XDR assay will complement the current Xpert assay, making it possible to select appropriate treatment (and infection control) approaches in patients identified with TB. The Xtend-XDR assay will use the same testing procedures and testing platform as the Xpert, and largely be restricted to patients who already test TB positive (and RIF resistant) in a primary Xpert test. These features will make it highly cost effective. We have developed proof of principal for all essential assay components. In the current proposal, we will finalize assay development, and create manufacturing processes, quality control, and assay software. Finally, we will perform the analytic and pre-clinical studies needed to prepare for clinical trials. These activities are exactly the type requested in the current partnership RFA. Our work will be performed in the following specific aims: 1. Finalize all assay parameters and internal control components in wet format. 2. Adapt assay for robotic assembly and modify assembly line for cartridge needs. 3. Develop analytic software and a simple interface for clinical operators. 4. Perform analytic and pre-clinical studies in preparation for clinical trials.